Journal: Pharmaceutics

Article Title: Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors

doi: 10.3390/pharmaceutics13050748

Figure Lengend Snippet: Overview of the five tested purification strategies to purify and to separate the empty and full recombinant adeno-associated virus (rAAV). For each experiment, only one harvesting method mentioned in the first arrow was performed. The abbreviations (in white) on the right side are also used throughout the text and refer here to the shown purification processes. AC, affinity chromatography; AEX, anion exchange chromatography; CEX, cation exchange chromatography; HIC, hydrophobic interaction chromatography; PEG, polyethylene glycol; TFF, tangential flow filtration.

Article Snippet: For some rAAV vector batches, the cell culture supernatant was harvested, filtered, and concentrated by tangential flow filtration (TFF) performed with a KrosFlo ® KRi2 universal using a 100-kDa mPES membrane from Repligen (Waltham, MA, USA) and buffered in 1× PBS (10-mM Na 2 HPO 4 , 1.8-mM KH 2 PO 4 , 4.27-mM KCl, and 137-mM NaCl, pH 7.4).

Techniques: Purification, Recombinant, Affinity Chromatography, Chromatography, Hydrophobic Interaction Chromatography, Filtration

Journal: Pharmaceutics

Article Title: Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors

doi: 10.3390/pharmaceutics13050748

Figure Lengend Snippet: Comparison of HIC-CEX-AEX and AC-AEX for purification of the rAAV2/8 vectors. Each chromatogram shows the respective ultra-violet (UV) signal in blue (scale on the left Y -axis) and the concentration of buffer B depicted as a dashed green line (scale on the right). ( A – C ) Chromatograms obtained with HIC-CEX-AEX in three subsequent steps on a HIC ( A ), a CEX ( B ), and an AEX ( C ) column. ( D ) SV-AUC analysis of rAAV2/8 of the indicated samples from different purification states. ( E , F ) Chromatograms obtained with AC-AEX in two subsequent steps on an AC ( E ) and an AEX ( F ) column. ( G ) Magnification view of the AEX chromatogram shown in F (focusing on the 120–150-mL range) to better visualize the peaks of the empty and full rAAVs. ( H ) SV-AUC analysis after AC and AEX. ( I ) Silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the rAAV material after TFF, and the intermediate steps from the purification downstream processes HIC-CEX-AEX and AC-AEX. AC, affinity chromatography; AEX, anion exchange chromatography; CEX, cation exchange chromatography; HIC, hydrophobic interaction chromatography; PEG, polyethylene glycol; rAAV, recombinant adeno-associated virus; TFF, tangential flow filtration; VP, viral protein.

Article Snippet: For some rAAV vector batches, the cell culture supernatant was harvested, filtered, and concentrated by tangential flow filtration (TFF) performed with a KrosFlo ® KRi2 universal using a 100-kDa mPES membrane from Repligen (Waltham, MA, USA) and buffered in 1× PBS (10-mM Na 2 HPO 4 , 1.8-mM KH 2 PO 4 , 4.27-mM KCl, and 137-mM NaCl, pH 7.4).

Techniques: Purification, Concentration Assay, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Affinity Chromatography, Chromatography, Hydrophobic Interaction Chromatography, Recombinant, Filtration

Journal: Pharmaceutics

Article Title: Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors

doi: 10.3390/pharmaceutics13050748

Figure Lengend Snippet: Effect of different harvesting methods on the rAAV2/8 vector production yield and pureness of the human embryonic kidney (HEK) 293T cell DNA . ( A – F ) Graphs showing the yield (total vector genomes, vg) for the rAAV2/8 vectors obtained with the two strategies, with materials harvested from ( A , B ) the cell culture supernatant and cell pellet lysate filtered, concentrated, and buffer-exchanged by tangential flow filtration (TFF) ( n = 3). ( C , D ) Culture supernatant by polyethylene glycol (PEG) precipitation ( C ), followed by HIC-CEX ( n = 2) and ( D ) followed by AC-AEX ( n = 5) or ( E , F ) cell pellet lysate ( E ) followed by HIC-CEX ( n = 2) and ( F ) followed by AC-AEX ( n = 5). ( G , H ) Graphs showing the removal efficiency of the strategies for HEK cell DNA at all the purification steps. AC, affinity chromatography; AEX, anion exchange chromatography; CEX, cation exchange chromatography; HIC, hydrophobic interaction chromatography; PEG, polyethylene glycol; rAAV, recombinant adeno-associated virus.

Article Snippet: For some rAAV vector batches, the cell culture supernatant was harvested, filtered, and concentrated by tangential flow filtration (TFF) performed with a KrosFlo ® KRi2 universal using a 100-kDa mPES membrane from Repligen (Waltham, MA, USA) and buffered in 1× PBS (10-mM Na 2 HPO 4 , 1.8-mM KH 2 PO 4 , 4.27-mM KCl, and 137-mM NaCl, pH 7.4).

Techniques: Plasmid Preparation, Cell Culture, Filtration, Purification, Affinity Chromatography, Chromatography, Hydrophobic Interaction Chromatography, Recombinant

Journal: Pharmaceutics

Article Title: Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors

doi: 10.3390/pharmaceutics13050748

Figure Lengend Snippet: Purification of the rAAV2/8 vectors using CEX-AEX(E/F). ( A – C ) Chromatograms obtained with a PEG-precipitated material and a CEX-AEX(E/F) process in only two steps on a CEX ( A ) and a novel CIMmultus PrimaS (AAV) ( B , C ) column. ( A ) CEX with a linear buffer B gradient. ( B ) CIMmultus PrimaS (AAV) with a linear buffer B gradient. ( C ) CIMmultus PrimaS (AAV) with a step gradient of 25%, 40%, and 55% buffer B. Each chromatogram shows the respective UV signal in blue (scale on the left Y -axis) and the concentration of buffer B depicted as a dashed green line (scale on the right). ( D ) Graph showing the yield (total vector genomes, vg) for the rAAV2/8 vectors obtained in the CEX-AEX(E/F) downstream process with material harvested by PEG precipitation ( n = 2) from the cell culture supernatant and cell pellet lysate ( n = 3). ( E ) Residual human embryonic kidney (HEK) cell DNA amount determined by real-time quantitative polymerase chain reaction (qPCR). ( F ) Silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the material from each step of the purification CEX-AEX(E/F) downstream process. AEX, anion exchange chromatography; CEX, cation exchange chromatography; PEG, polyethylene glycol; rAAV, recombinant adeno-associated virus; TFF, tangential flow filtration; VP, viral protein.

Article Snippet: For some rAAV vector batches, the cell culture supernatant was harvested, filtered, and concentrated by tangential flow filtration (TFF) performed with a KrosFlo ® KRi2 universal using a 100-kDa mPES membrane from Repligen (Waltham, MA, USA) and buffered in 1× PBS (10-mM Na 2 HPO 4 , 1.8-mM KH 2 PO 4 , 4.27-mM KCl, and 137-mM NaCl, pH 7.4).

Techniques: Purification, Concentration Assay, Plasmid Preparation, Cell Culture, Real-time Polymerase Chain Reaction, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Chromatography, Recombinant, Filtration